Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2-->8)-polysialic acid

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Evidence for the extended helical nature of polysaccharide epitopes.

The 2.8 A resolution structure and thermodynamics of ligand binding of

an antigen binding fragment specific for alpha-(2-->8)-polysialic acid

Evans, S.; Sigurskjold, B. W.; Jennings, H. J.; Brisson, J.-R.; To, R.; Tse, W.

C.; Altman, Eleonora; Frosch, M.; Weisgerber, C.; Kratzin, H. D.; Klebert, S.;

Vaesen, M.; Bitter-Suermann, D.; Rose, D. R.; Young, N. M.; Bundle, D. R.

Evidence

for

the

Extended

Helical

Nature

of

_{Polysaccharide Epitopes. The}

2.8

A

Resolution Structure

and

_{Thermodynamics}

of

_Ligand

_Binding

of

an

_Antigen

Binding

Fragment

Specific

for

a-(2—1'8)-Polysialic

Acid*

*

S. V. Evans,*°° v

B. _{W. Sigurskjold,v}§ _{H. J.}

Jennings,*v J.-R. Brisson,vR. To,v W. C. Tse,v E. Altman,v

M.

Frosch,11 C. Weisgerber,v H. D. Kratzin,A S. _{Klebert,A M. Vaesen,A} D. Bitter-Suermann,11 D. R.

Rose,vl

N. _{M. Young,v} andD. R. Bundlev’#

Institute

_for

_BiologicalSciences, NationalResearchCouncil

_of

Canada, Ottawa, Canada K1A 0R6, Max-PlanckInstitutfur

Experimentelle Medizin,AbteilungImmunchemie, 37075 Gottingen, Germany, and Medizinische Hochschule Hannover, Institut_{furMedizinische Mikrobiologie,} 30625 Hannover, Germany

Received_May23, 1994; RevisedManuscriptReceivedNovember9, 1994®

abstract: _{The antigen binding fragment}

from

an _IgG2a k murine monoclonal antibody

with

_specificity

for

a-(2—1•8)-linked sialic acid polymers has _{been prepared and crystallized in} the absence

of

_hapten.

Crystals were _{grown by vapor}

diffusion equilibrium with

16—18% _polyethylene

_glycol

4000 solutions.

The structure was solved _{by molecular replacement methods and} refined to a conventionalR factor

of

0.164

for

data to 2.8 A. The _binding siteisobserved to _display a _shapeand distribution

of

_{charges that}

is_{complementary}tothat

of

the_{predicted conformation}

of

the_{oligosaccharideepitope.}

A

_{thermodynamic}

description

of

ligand binding has been _compiled

for

_{oligosaccharides ranging}

in

_length

from

9 to 41

residues, and the data

for

the largest ligand has been used in a _{novel way} to estimate the size

of

the antigenbinding site.

A

model

of

_{antigenbinding} is _presentedthat satisfies thisthermodynamicdata, as

well

as a _{previously reported requirement}

of

conformational specificity

of

_{the oligosaccharide.}

_X-ray

crystallographic and thermodynamic evidence are consistent

with

a _binding site that accommodates at

least _{eight sialic} acid residues.

The capsular polysaccharides

of

_group B meningococci and Escherichia coli capsular type

Kl,

which both cause

human meningitis, consist

of

a_homopolymer of

a-(2—*8)-linked_{sialic acid (V-acetylneuraminic acid;Jennings et}al.,

1984). The polymer is known to be _{poorly immunogenic}

(Wyleet al., 1972; Jennings & Lugowski, 1981). That this

poor immunogenicity is due to molecular_mimicry is

sup-ported bytheidentificationof_{structurally identical}

a-(2—8)-linked sialooligosaccharidesinhumanandanimalfetaltissue (Finne et al., 1983). Chains

of

a-(2—8)-linkedsialic acid

are _{developmental antigens (Rutishauser}

&

Jessel, 1988a),

are carried by glycoproteinsinvolvedinneuralcelladhesion

(Rutishauseret_{al., 1988b;Phillips}etal., 1990; Brandleyet

al., 1990; Corralet al., 1990; Berg et al., 1991), and have also beenidentifiedin othertissues_includinghumantumors (Troy, 1992). Despite its poor antigenicity, antibodies to a-(2—1"8)-polysialicacidcan be_producedin _special circum-stances. One suchmonoclonal antibody (IgG2a,designated

*

Coordinateshave been depositedwith theProtein Data Bank at

BrookhavenNational Laboratories,code 1PLG.

*

Correspondingauthors.

"

Present address:_DepartmentofBiochemistry,UniversityofOttawa, 451 _{Smyth, Ottawa,} CanadaK1H 8M5.

v_{National ResearchCouncilofCanada.}

§_{Present address:}

DepartmentofChemistry, Carlsberg Laboratory,

Valby, Copenhagen DK-2500, Denmark.

a_{Max-PlanckInstitutfiir}

Experimentelle Medizin.

11Medizinische Hochschule Hannover.

1_{Present address: Ontario Cancer Institute and}

Department of Medical Biophysics, University of Toronto, 500 Sherboume St.,

Toronto, Ontario,Canada M4X 1K9.

* _{Present address:}

University ofAlberta, DepartmentofChemistry, Edmonton, Alberta, CanadaT6G2G2.

8

Abstract published in Advance ACS Abstracts, February15, 1995.

mAb735)has beencharacterized (Froschetal., 1985), and its heavy and_{lightchains have been sequenced (Vaesen et} al., 1991; Klebertet al., 1993).

This antibody, itsFab,1 andothera-(2—1"8)-polysialicacid specific antibodiesallrequirean _{unusuallylong segment}

of

haptenforbinding, and the_{affinityappears to increase}with

increasing chain length(Jennings et al., 1984, 1985; Finne

&

Makela, 1985;Rabatet_{al., 1988;Hayrinen}etal., 1989).

It has been proposed that antibodies recognize a _specific

helical conformation

of

this polysaccharideandaminimum

of

10 residues is _required to form a helical epitope. The

validity

of

this propositionwas further strengthenedby the

identification of a common _{length-dependent epitope}

re-sponsible forthe cross-reaction

of

a-(2—8)-polysialic acid

and _{poly(A) with} a human monoclonal macroglobulin

(IgMN0V; Rabat et_{al., 1988) and the known propensity}

of

poly(A) to form helices of n = _8—10

(Yanthindra

&

Sundaralingam, 1976). Thisconformationaldependenceon

chain lengthhas been _{confirmed by NMR (Michon} et al., 1987), and similar chain length-dependent conformational epitopes have been described for other antigenic capsular polysaccharides (Wessels et al., 1987; Wessels & Rasper, 1989). Bymeans

of

_{potentialenergy}calculationsandNMR, it was _{shown that, although mostly random}coil in nature, a-(2—1-8)-polysialic acidcan _{adopt extended helical}

confor-mations where n ^ 9 (Brisson et al., 1992), the stability

1

Abbreviations:ELISA,enzyme-linked immunosorbentassay; Fab,

antigenbinding fragment; PEG,polyethyleneglycol;LI,L2, L3, HI,

H2, and H3,the _{six hypervariable loops} as _{defined by Kabat} et al.

(1991);HEPES,AL(2-hydroxyethyl)piperazine-Y-2-ethanesulfonicacid;

SDS—PAGE, sodium dodecyl sulfate-polyacrylamide gel

electro-phoresis.

0006-2960/95/0434-6737$09.00/0 Published 1995 _by the_{American Chemical Society}

Downloaded by NATL RESEARCH COUNCIL CANADA at 11:57:01:390 on July 05, 2019

6738 Biochemistry, Vol. 34, No. 20, 1995 Evans et al.

ofwhichare _dependenton its_{carboxylategroups}(Baumann etal., 1993). Ithas also beenshownthat_{mAb735 displays}

a_{fairlyhighdegree}of_specificityfor_{the 77-acetylneuraminic}

acid polymer,asthereis_onlya_{small cross-reactivity (%5%)}

observed with the _77-propyl analogue (Jennings et al., unpublished results).

The determination

of

the three-dimensional structure

of

the_{antigen binding fragment}anda_{thermodynamic} descrip-tion

of

_{the hapten binding}

of

such a _system are _necessary

steps in extending our _{understanding}

of

the interactions between antibodies andspecific conformations

of

antigenic polysaccharides. We now _report the _preparation,

thermo-dynamic characterization, crystallization, molecular replace-ment solution, and2.8

A

resolution structure

of

aFabfrom

the murine _{monoclonal antibody mAb735 specific} for

a-(2—8)-linked polysialicacid. We_{also present the}results

of

_modeling studies on the nature

of

the _{antibody-antigen} complex carried out _using the structure

of

the_binding site reported herein and the structure

of

the antigenpreviously reportedfromNMR experiments (Brisson etal., 1992).

EXPERIMENTAL

PROCEDURES

Preparation

of

Fab. TheFab was _{prepared by digesting}

30.4 mg

of

mAb735 (IgG2a) in 15 mL

of

10mM Tris-HCl

containing 50 mM dithiothreitol _{(pH 8.0)} with 152 _pg

of

papain (IgG:papain = _200:1) for ₅ h at _room

temperature. The solutionwas _dialyzed at4 °C_{against three changes}

of

10 mM _{Tris'HCl (pH} 8.0) and applied to a DE52 ion

exchange column. TheFab fragmentwas eluted with the

same buffer. SDS—PAGE

of

the_purifiedFabunder

reduc-ing conditions showed two bands

of

_apparent molecular weight30and26_{kDa, corresponding}to theFdandLchains

of

the Fab, andisoelectric focusing showeda_{single band}at

p/

8.4.

Preparation

of

Oligomers

of

Sialic Acid. Oligomers

of

sialic acidwere _{obtained by partial acid hydrolysis}

of

the sodium salt

of

colominic _{acid purchased} from Nacalai Tesque, Inc. (Kyoto,Japan); 100mg

of

colominic acidwas

dissolved in 3 mL

of

water, the_pH brought down to 2.0, and_{the sample heated to 80} °Cfor 1 h. After_cooling,the

pH was _{adjusted to 7.0, and crude} fractions

of

_varying

molecular weight withchain lengths ranging from 9 to 15

were obtainedfrom BioGel P-10,

200-400

mesh (BioRad Laboratories) chromatography eluted with 30 mM

am-monium bicarbonate buffer, pH 8.0. The fractions were

dialyzedagainst water andlyophilized. Furtherpurification was carried out _{by preparative exchange chromatography}

(Dionex anion) using a Carbo-Pac PA1 (9 x 250 _mm)

column with _{pulsed amperometric detection. The eluant}

consistedof0.15 M_{sodium hydroxide}witha_gradientfrom

50 mM to 1 M sodium acetate (followed by _post column

additionof0.5M_{sodium hydroxide).} After_{passagethrough}

thedetector,theeluantfractionswere _{immediately adjusted}

to _pH7.0withacetic acid. A_{homogeneouspreparation}

of

colominic _{acid corresponding} to an _{approximately} 12 kDa

polymer (equivalent to _{approximately} 41 _residues) was

obtained as the _{highest molecular weight} fraction from

Sephadex G-100 chromatography

of

the sodium salt

of

colominic acid using 10 mM sodium _{phosphate, 0.15} M

sodium chloride, pH 7.2.

Microcalorimetry. The_{thermodynamicparameters}forthe

binding of the a-(2—8)-linked sialic acid polymer by the

mAb735 Fab were obtained by titration _{calorimetry using}

an OMEGA titrationmicrocalorimeterfrom MicroCalInc.,

(Northampton,

MA)

(Wisemanetal., 1989). Asolution

of

76 /<M Fab in 10

_pM

_{sodium potassium phosphate}buffer

containing0.15 M _{sodium chloride, pH}7.4, was titratedat

25 °Cwith_ligandinthesame _{buffer. Twenty injections}of

5

_pL

froma100

_pL

_{syringe stirring}at_{400 rpm}were carried out from a solution

of

13.35 _{mg/mL ligand.} The thermo-dynamics

of

thebinding ofoligomers to _{mAb735 IgG} was

determinedinthe same _{way. Dataanalysis}

of

the _binding isotherms was carried out as _{described previously} (Sigur-skjoldet al., 1991).

Crystallization andX-rayDiffractionAnalysis. Crystals

of

mAb735were _{obtained by}the_{hanging drop method.}The reservoir solutionwas _composed

of

16—18%PEG-4000,0.1

M HEPES at_pH 7.5, 0.1M (NH4)2S04, and 0.01 M NaN3. The_drops were madeby mixing6.0

pL of

6.7 _mg/mLFab

with6.0

_{pL of}

reservoir solutionon thecoverslipandseeded

1 _{day later} with _{microcrystals grown previously. Crystals}

appearedafter1 weekand_{grew to}an _{average size}

of

0.3 x

0.3 x 0.4 mm3. _Crystals

of

mAb735are orthorhombic,with

a = _79.14

k,b

= _{91.21 A, andc} = _141.4 A.

Systematic

absencesshowed the_spacegrouptobeeither1222oxI2\2\2\, withone _{molecule per asymmetric unit.}

X-ray diffractiondatafroma_{singlecrystal}were collected

on aSan_Diegomultiwire_systemtwo-detector_setupmounted

on a_{Rigaku RU-200 rotating}anode_{X-raygenerator operated} at 40kV and 120mA. The detectors were _positionedat 6 values

of -12.0°

and30.0°, and thediffraction sphere was

sampledin seven _sweeps. A total

of

87625 measurements

were made

of

10590 _{unique reflections,} out

of

a total

of

10611 _{possible reflections, yielding} a data set 99.8% complete to 2.8

A

resolution. No explicit corrections for

absorptionor _decay were _applied.

Molecular _{Replacement Solution.} The structure was

solved in _{space group} 1222 _{via molecular replacement}

methods. Solution_proceededintwo _{stages, using}the

anti-Brucellaabortus YsT9.1 Fab _{structure (Rose et} al., 1993)

as a_{model. First,}theorientationand_position

of

theconstant regionwere _{determined using the}MERLOTpackage (Fitzger-ald, 1988). Many combinations

of

resolution range and a

cutoffwere tried, with the cleanest results obtained using

datain _{the range 6.0—4.0}

A

resolution with/ > _3o(l).

The positioning

of

the variable region was less straight-forward,as no combination

of

_{data range and}a cutoff_using

MERLOT led to a solution. The correct _position of this

fragmentwas locatedvia a_packingserach_utilizingthenow

correctly positionedconstant_{region, by allowing}thevariable regiontorotateabout the Fab ‘hinge’residuesin2°intervals,

while_{monitoring all intra-}andintermolecularcontacts. This

procedure definedan allowed sectionof_{approximately} 15°

ofarc inwhichthevariable region couldbeaccommodated bythecrystal packing. However,asix-dimensional_{fine grid} search about the allowed section using the entire variable region

of

the YsT9.1 Fab model did not _yielda match to the observed diffraction _{pattern. The} final molecular

re-placement solutionrequiredthe separate positioning ofthe heavy and light chains of the variable region

of

the Fab, usingtheprogramBRUTE(Fujinagaand Read, 1987),with

theentire constant _regioninputas a _stationary atoms set.

All

refinementswere carriedoutwiththe_programXPLOR

(Briingeret al., 1988)usingdataintheresolutionrange6.0—

Specific for a-(2-*8)-Polysialic Biochemistry, Vol. 34, No. 20, 1995 6739

Figure 1: Isotherm for the _binding of the Fab to the 12 kDa a-(2—*8)-linked sialic acid _oligomer. The dottedline shows the calculatedfit_(Sigurskjoldet_{al., 1991) assuming that two Fabs}bind

each _{41-mer oligosaccharide.}

/

theYsT9.1 model. _{Rigidbody refinement}

of

themolecular replacement solution

of

the mAb735 began with just the variable and constant _{regions defined} as _rigid bodies and thenwitheach

of

thosedivided into heavyand_lightchains.

Afteratotal

of

50_cycles,theR_{factor dropped}to 0.33from

the

initial

0.48. Refinement_proceededwithseveralrounds

of

simulated annealingwith an

initial

_temperature

of

3500 K. Conventional positional refinement with an overall temperature factor applied further reduced the R factor to 0.26 after 50 _cycles. After a _{single round}

of

manual

intervention, refinement proceeded again using an overall

temperature factor to yield an R factor

of

0.24 after an

additional50 cycles. To avoid biasingthe struture,no water molecules were included intherefinement.

At this _{point in} refinement, the sequences for both the

heavyandlightchains becameknown (Vaesenet _al., 1991; Klebertetal.,1993) andwere _imposedon the structure. The

final set

of

refinements includedtheplacement

of

41 water

molecules and individual _{atomic temperature factors} were

allowedtorefine. Convergencewas reached afteratotal

of

50_cycles to_yieldafinalRfactor

of

0.164 ataresolution

of

2.8 A.

RESULTS

AND

DISCUSSION

Thermodynamics. Three parameterscan beobtainedfrom

nonlinear regression analysis ofthe calorimetric isotherm: the_{molar enthalpy}(AH°),the_bindingconstant _(K),and the apparent concentration

of

binding sites (Fab) (Wiseman et

al., 1989; Sigurskjoldet_{al., 1991), and} fromthese values,

AG° and AS° can be calculated. The isotherm for the bindingoftheFab to the 12kDaa-(2—8)-linkedsialic acid polymer consisting ofapproximately 41 residues is shown in Figure 1. _{(Unless otherwise} stated, all figures were

generatedwiththeSETOR programpackage; Evans, 1993.) This isotherm is a _{smooth sigmoid curve,} i.e., it describes

only a _{singlebinding} constant. This means that

if

thereis

more than one _epitope on _{this polysaccharide chain,} then

the _binding

of

one Fab to the _{polysaccharide} does not

significantly facilitate (nor debilitate) the _binding

of

a

subsequent Fab. Such cooperativity in binding has been observed, e.g. , for the type

III

group B Streptococcus

capsularpolysaccharide(Wessels et al., 1987). Theisotherm

was fitted four_{times assuming that} the_polymercontained

either one, two, three, or four _epitopes for _binding to the Fab,respectively,and the apparentconcentration

of

Fabwas

determinedineachfit. _{The regression}resultsare shownin

Table 1, andonly thevalue

of

73

_pM

_{Fab—corresponding} to two epitopeson theligand—is consistentwiththeknown concentration

of

76 Fab. Itisconcluded thata_polymer

of

_{approximately}41 sialic acidresidues can accommodate

only two Fab molecules. The calorimetric measurements therefore provide an estimate

of

the extent to which an

individual_{Fab renders adjacent segments}

of

the_{antigen chain}

unavailablefor_binding. Inthiscase,themaximum number

of

sialic acidresidues involvedis from 15 to 20, anumber thatis consistent with a_binding site around 10residues in

length. This also demonstrates anovel use

of

_calorimetry

to determinethe size

of

a_{binding epitope. The association}

is_{enthalpy driven}witha_largeunfavorablechangeinentropy. Strong enthalpic interactions combined with unfavorable entropychangesare _{usually observed}for

protein—carbohy-drateinteractionswith_{longsugarchain lengths[Sigurskjold}

et al. (1991) andreferences therein].

For comparisonthethermodynamics

of

the_binding

of

the whole mAb735 IgGto _oligomers from 9 to 15 residuesin

lengthare shownin Table 2. The_binding

of

these _ligands (Ka ~ 4 _x 104M-1) is ₁ order of _{magnitude weaker}than

forthe_{long chain (Ka} ^ 3 x 105_M-1)- Thereseems tobe

little

or no _{chain lengthdependence} for_any

of

the thermo-dynamic functions in this range

of

chain lengths, which

seems to contrast with _{previous observations (Jennings et}

al., 1984, 1985; Finne

&

Makela, 1985; Rabatetal., 1988; Hayrinenet_{al., 1989);however, this trend may}beconcealed by thelarge standard deviations observedfor —TAS°. We

can observe a _{relatively strong chain length dependence}

between _binding

of

the _{long polymer} and the shorter oligomers. Theenthalpy

of

bindingforthelongpolymeris twice that

of

the_{shorter ligands. Thus}it seems thattheFab is able to take full _advantage

of

the _{enthalpic interactions}

for the _{whole epitope}

of

about 20 residues. The entropy change, on the otherhand, has becomeprogressively more

unfavorable, —TAS° _being 3 _{times greater}for the_polymer than for the shorter oligomers and thus compensating for

some

of

the _{enthalpy gain. This reflects that—as} expec-ted—thereisa_{strongentropypenalty inbinding longer}chains

(if

—TAS° for the _polymer were _just twice that of the oligomers, K wouldbeabout 109

_{M_l).}

X-ray Crystal Structure Analysis. A summary of the

deviations between stereochemical_parametersderivedfrom

the final _cycle of refinement with ideal values (Table 3)

shows that _{this structure displays geometry that}is in _good

agreement with literature values and has all non-glycine residues,withthe_exception

of

V56on the _lightchain,inor near the_{allowed regions}ofa_{Ramachandran plot, Figure}2.

Additionally,examinationover thecourse ofthepolypeptide

chain shows that, with a few exceptions distant from the bindingsite,all main chainand sidechain atomsare observed to _{occupy appropriate electron density. Figure} 3 is an

electron densitymapofthe_heavychain

HI,

which illustrates

the well-defined density that is _{typical for} residues in the

6740 Biochemistry, Vol. 34, No. 20, 1995 Evans et al.

Table 1: ThermodynamicsandDeterminationofEpitopeSizeofthe_Bindingby mAb735 Fabofa 12kDa Polymerof a-(2—8)-Linked Sialic

Acid_{(Corresponding}toApproximately 41 Residues)Obtained byTitration_{Microcalorimetry0}

assumedno. of_epitopes K (xlO-5M-1) AG° (kJM"1) AH° (kJM-1) —7AS° (kJM-1) apparentcone ofFab_(«M) 1 7.3±4.0* —33.8±1.4 — 161.6± 15.1 127.8± 15.8 37±4 2 3.4± 2.0 —31.9±1.5 -81.6±8.5 49.8±9.3 73± 8 3 2.4± 1.4 —31.0± 1.4 -54.1 ±5.3 23.1 ±6.1 110± 13 4 1.7±0.9 -30.1 ± 1.4 -41.0 ±4.1 10.9±4.9 146± 17 0_{The concentrationof}

mAb735Fabinthe_experimentwas 76wM.b_{Uncertainties}

represent three standarddeviations.

Table2: _{Chain Length Dependence}ofthe_{Binding of}

a-(2—8)-Linked SialicAcid_Oligomersto_{mAb735 IgG Obtained}

byTitration Microcalorimetry chain K AG° AH° -TAS° length (x10-4M_l) (kJM"1) (kJM-1) (klM”1) 9 3.9± 0.9° -26.2 ± 0.6 -45.4 ± 6.7 19.1± 6.8 11 4.8± 0.9 -26.7± 0.5 -42.6 ± 2.9 15.8± 2.9 14 5.9± 2.9 -27.2 ± 1.2 -38.7± 2.8 11.5± 3.1 15 4.4± 1.9 -26.5 ± 1.1 -41.6 ± 6.8 15.9± 7.3 average 4.4± 0.9 -26.6±0.5 -41.0 ± 2.6 14.4± 2.7 “_{Uncertainties}

represent three standard deviations.

Table3: _{Stereochemistry}oftheFabfrommAb735 after Refinementto 2.8AResolution

stereochemical rms deviation refinement restraint refinement parameter fromidealvalues _{weighting parameter}

bonddistances(A) 0.014 0.030

angledistances(A) 0.052 0.040 planar1—4distances(A) 0.065 0.060

planes(A) 0.022 0.030

chiral volumes(A3) 0.125 0.125

cudihedral_angles(deg) 3.5 15.0

single dihedral contacts(A) 0.232 0.250

multipledihedralcontacts _(A) 0.201 0.250

-120 - ^

o

-180 MI

ytt

U--U-J-,-i

-180 -120 -60 0 60 120 180

9

Figure 2: Plotofmain chain dihedral_angles (Ramakrishnan &

Ramachandran, 1965)ofall non-glycine residuesofmAb735. The_{framework regions}ofthe_{molecule adopt} conforma-tions similar to those _reported for other Fabs and _display theusual_{immunoglobulin}fold withan elbow_anglebetween the_{pseudo-2-fold}axes

of

thevariableandconstant domain

dimers_{equal to 166°. The}moleculesare _packedinthe usual head-to-tail fashion observed formost Fabs, withthe _long axis

of

the _{molecule nearly coincident} with the unit cell

c-axis. Seven

of

the 15 observed intermolecular contacts

are betweenresiduesinthe_{hypervariable regions}and the C domain dimers

of

adjacentmolecules, mostlyinvolvingthe

LI

andHI _loops.

Asthe_{shapeand charge}distribution

of

the_{oligosaccharide} are of _{paramount importance} in _{describing its antigenic}

properties, the binding surface

of

the Fab is also best described in that manner. _{The antigen binding} surface is bimodal in that

it

_undergoesa_strikingreversalin_{shape and}

charge distribution along the interface between the heavy

and_lightchains. Theleft side

of

thesite(asinFigure4) is

where L3, H2, and the _‘tip’

of

H3 are observedto form a

convex surfacewhich_{displays threepositivelychargedlysine} residues,Lys H59,LysH65, and_{Lys H101. Theright}side

of

the site finds L2 and thebulk

of

_{H3 meeting}to form a

groovewhichhas a_{relativelyuncharged}surface_exceptfor

the_{negatively chargedAsp}

HI05

residue at thebottom

of

the_{groove, with positively charged Arg} H98_just above

it

on the side. _Loops

LI

andHI _display no _{charged residues}

except Asp H31, which is well away from the observed groove.

Modelingthe_{Hapten into} the_BindingSite. _X-ray crystal-lographicstudiesof_complexes

of

Fabswith_{oligosaccharides}

have shown that the _specificity

of

the binding site on the Fabs_{usuallyencompasses}one tofoursugar residues (Cygler etal. 1991;Oomenetal., 1991;Vyasetal., 1993);however, thereare severalreasons _{to suppose} thatthebinding siteon

mAb735 is _{much larger. Firstly, binding} studies have demonstrated that a critical _{chain length}

of

10

a-(2—8)-linkedsialic acidresidues isrequiredinorderforthe hapten

tobindto_{mAb735 (Hayrinenet al., 1989), and}thishasbeen confirmedinour _{thermodynamicanalysis. Secondly,}NMR

studies _{in conjunction} with _{potential energy} calculations (Brissonet_{al., 1992)} are consistentwith_{the presence}

of

an

extendedhelical_{epitopein a-(2—8)-polysialic}acid. _Thirdly,

from _examples where _binding studies have been _reported (Jennings etal., 1985; Finne

&

Makela, 1985;Kabatet_al.,

1988; Hayrinen et al., 1989), all antibodies specific for

a-(2—8)-polysialic acid reported to date are based on an

extended_{helical epitope. None}have been detected with a

specificityforshortera-(2~*8)-sialooligosaccharides. Thus, anymodelofhaptenboundin the _binding site of mAb735

must reflectthis helicalnature ofthe antigenicformofthe

hapten andallowcontactbetweenantibodyand antigen along

a_spanthatis

of

theorder

of

10residuesinlength. Finally,

themodel must _explainthe_specificity thatmAb735 shows

forthe_A-acetyl-vs _{N-propylneuraminic}acid. Althoughthe

'H

NMR _{analysis provides overwhelming evidence} for the requirement of a helical conformation for the _antigen, the

Specific for a-(2—8)-Polysialic Biochemistry,

Figure 3: Stereoview of the _tip of the H3 loop (thick lines) and surrounding residues, showing the corresponding observed electron density (thin lines) plottedat a 1.5<jlevel. With few_exceptions, all residues in the _{protein display excellent} agreement withobserved electron density.

Figure4: Stereoviewofthe_antigenbindingsurface_(Connolly, 1981)ofmAb735 looking down the_{pseudo-2-fold}axis. Those _portions

ofthe_{surface due to charged atoms} are shown withdenser dotdistributionand havetheir_{parent residues labeled.}

Figure5: Side and top views withrespect to the helixaxis for helical modelsof_{a-(2—8)-linked sialic}acid 10-mers constructed

to fit 'H NMR solutiondata(Brisson et

_al„

1992). Although the

pitchofthe helixthatcan beaccommodated from the NMRdata

can _{vary significantly, all helix models require the negatively} chargedcarboxylicacid groupsto facetheinteriorofthehelixwhile

the N-acetyl groups face the exterior ofthe helix. The index n

showsthenumberofresidues perturn foreachhelix.

exact _pitchofthehelix thatcan be_generatedto fit thisdata

can _{vary significantly. Figure} 5.

Flelicesare definedin terms ofaset

of

helical _parameters (n,h) with n _being the number of _{residues per} turn and h

being the translation along the helix axis ofcorresponding residues. The pitch is defined as n x h. Helices were

generated as described before (Brisson et

al„

1992). The rise per residue, h, forthe n= ₆helix _{was 6.0A,and5.8}A

for the ;i = ₉ helix. Extended helices

of

nearly identical energy can be _generated forn = _6—11

by a _slightchange

(30°) in _only one

of

the four dihedral _{angles linking} two residues (Brisson et al., 1992). For the n = _{6and n} = ₉

helix, this dihedralanglediffers by only 20°. This demon-strates that the _pitch

of

these extended helices can _vary

greatly by minimal rotation about only one bond and with

very little change in energy.

All

of

thesehelicalmodels

of

_{polysialic acid}have several importantfeatures in common. Most_{significantly,} in each

model the _{negatively charged carboxylic acid groups face}

the interior of the helix with a _{repeat distance}

of

5—8 A

whilethe (V-acetylgroupsface theexteriorofthehelix. This

means thatfor_any

of

themodels,over one turn ofthehelix,

one endofthe_{oligomer wouldpresent thenegativelycharged}

carboxylic acid groups to the _binding site on the antibody

while the _midpoint of the _{oligomer would present the}

relatively uncharged but protruding/V-acetyl groups. This reversal

of

environment fromone endofthe _epitope to the other is themirrortothat described abovefortheFab_binding site, whereone extreme

of

the_{bindingsite presents} aseries

of_{positivelychargedlysine groups}with thecorrect _spacing

to form salt_bridges with the _{carboxylicacid groups} on the antigen, whiletheotherextreme ofthe_{bindingsite presents}

6742 Biochemistry, Vol. 34, No. 20, 1995 Evans et al.

Table4: SuccessiveRotationofSialicAcidResiduesinthe Model of_{Bound Hapten}

sialic acidresidues rotation (deg)

2-3 66.5 3-4 58.0 4-5 74.5 5-6 50.3 6-7 72.3 7-8 68.2 8-9 52.6

a_{groove which}can accommodate and_{recognizethe IV-acetyl} groups.

The observation

of

_{shape and charge complementarity}

between antibodyand_{antigenstronglysuggests}amodelfor

mAb735—polysialic acid binding, and unlike the helix

modelsderivedfromthe

NMR

data, thebinding siteon the Fabhasvery definite dimensionsandcan _onlyaccommodate

a.narrow _range

of

helix_pitch. In order for _{the hapten to}

formboththe_{salt bridges and beproperly oriented to project} the_{IV-acetylgroups}intothe_{observed groove}on the surface

of

the Fab, the oligosaccharide helix must make

ap-proximately V2

of

aturn within 17 A. This_{corresponds to}

helixa

of

_Figure5,withn = _6residues

perturn anda_pitch

of

n x h = ₃₆A.

The _approach taken for _modeling the _complex was

conservative. No main chain atoms

of

the _protein were

moved. Sidechain conformations forboththe_proteinand theoligosaccharidewere allowedto _{vary betweensterically} favoredrotamers. Inorderto best_comparethe

fit

between oligosaccharide model and Fab, the basic helical shape

of

the searchmodelwas _{preserved, and}Table4 showsthatthe angle

of

rotationbetween each successivepair

of

sugarrings is close to the 60° _expectedforan n = ₆helix. However,

small changes inthedihedral angles ofthelinkingregions

of

the _{oligosaccharide} were _required to _bring suitable hydrogen-bondingpartnersinto proximity. TheNMRdata

will

accommodate a _range

of

_pitch for the _{oligosacchride} links, andalthough themodelsthatare _presentedin_Figure 5containlinks

of

identical pitch,anymodelcan containlinks

of

different _pitch. Two water molecules that had been

located inthe_binding site_duringtherefinement procedure

were movedlessthan 1.0

A

suchthatthe_original hydrogen-bonding partner was maintained. A third water molecule,

Table5: Numberand_Typeof_{Hydrogen Bonds Found}on

OligosaccharideResiduesintheModel

sialic acidresidue total toCOO to_A-acetyl

2 3 0 3 3 7 0 2 4 5 0 3 5 4 2 1 6 4 1° 0 7 2 1 0 8 2 1“ 1 9 2 1“ 0 “

Chargedresidueinteraction.

Wat-21, whichwas inthe second_hydrationshell, had tobe

removed tomake_way forthe _{oligosaccharide.}

The

initial fit

of

the 10-merhelix in _Figure 5ais shown

in_Figure6. Thethree_predictedsalt_bridgeswiththe_lysine residuesare_{easily formed}withsmall_changesinthedihedral angles

of

thelinking regions

of

the oligomer, andthe turn

of

the helix _{is adequate to bring} an _{appropriate A-acetyl}

group as well as much

of

its parent sugar residueinto the groove observedon theface

of

the Fab. _{The highdegree}

of

complementarityofthe

initial

fit of

thehelixto the _binding surface

of

theFabis evident,aseachone of_{eight consecutive}

residues of the oligosaccharide makes _{significant overall} contact with the protein, forming at least two _hydrogen bonds,Table5and_Figure7. Table5alsoreflectsthehelical nature

of

the_{antigen, where}thefirstresiduesin _{the hapten}

are bound _{chiefly through} the _{A-acetyl groups,} while the latterresidues form salt_bridges to the carboxylate anions.

Withthe_exception

of

LI,

each

of

the_{hypervariable loops}

can make at least one _{hydrogen bond directly with} the modeled _hapten; however, residues on LI can serve to stabilize the _position of a water molecule in a _position to

forma_{strongbridgeto the hapten,Figure}7. No_significant contact is madebetweentheframeworkresidues oftheFab and the oligosaccharide model. Table5 and_Figure 7 both showthatthemodel

of

the _antigenic form

of

the oligosac-charide can make as _many as 30 _hydrogen bonds withthe Fab,withaAH°

of

about —41kJM-1. This_compareswell with the crystal structure and _{thermodynamics (Bundle} et al., 1994) of the_{Salmonella O-polysaccharide antigen} Fab complex (Cygler et _{al., 1991),} which shows 12 _hydrogen

bonds with aAH°

of

about —

20 kJ M_1.

Figure 6: Stereoviewofthe fit ofthehelical modelofa-(2—8)-linked sialic acid to the_binding surfaceofFabmAb735. The Fab is

viewed approximately downits_{pseudo-2-fold}axis,withthe_lightchainpositionedabove and the heavy chainbelow _{the hapten. The}left

sideofthe_diagramshows the salt bridgesformedbetween three surfacelysinegroups and thecorrespondingcarboxylicacid groupson the hapten. Theright sideshowsthe contact oftheA-acetyl groupsofthe haptenwith surfacefeatureson the Fab.

A

Fab_{Specific for a-(2—8)-Polysialic} acid _{Biochemistry,} Vol. 34, No. 20, 1995 6743

Figure7: Stereoview ofthe_specificcontacts made_byan _{eight-residuesegment}oftheoligosaccharideshownin Figure5ato the surface

ofthe Fab,showingthe_highdegreeofcomplementarilyofshapeand charge between theprotein and carbohydrate. Modeled hydrogen

bondsare shownwithdashedlines. Theproteinresiduesare labeledwiththeir one-letter amino acidcode and toindicatetheir_parentlight (L)or _heavy (H) chain. The _{oligosaccharide}residuesrun fromSia-2_(top) toSia-9 (bottom).

The greatestnumber

of

_{interactions between protein}and hapten are found alongthe surface_grooveon the_rightside

of

theFabas seen in_Figure4 and_involvingSia-2—Sia-5. It

isin thisarea thatthe_specificity

of

mAb735_{for A-acetyl}vs

A-propyl polysialic acid becomes apparent, as a series

of

hydrogen bonds is formed between several sialic acid residues and thebottom and sides

of

the _{groove that}would

not be_possibleforthelongerA-propylgroup. OneA-acetyl group (on Sia-3) forms a _{hydrogen bond} with _{Asp H105.}

ResiduesSia-6—Sia-9are _{primarilyinvolved through their}

respective carboxylic acid _groups, which take _part in one

hydrogen bond, and threesalt bridgeswith lysine residues

on the _{protein. There} are at least six additional hydrogen bonds thatcan be_readily modeled betweenthe_protein and other atoms

of

the sugar, butthe formation

of

salt _bridges between the_protein and the_{carboxylic acid groups} of the sialooligosaccharidesisan _{important feature}inthe

recogni-tion

of

the_{helix,and compares}well withthecrystalstructure

of

the_complex

of

asialic acid_analoguewithinfluenza virus

neuraminidase (Vargheseetal., 1992). Itisin thisarea that the_{slight cross-reactivity}ofmAb735withthe_A-acetyland

A-propylneuraminic acid couldreside.

Althoughthe chargedistributionon the_proteinis the most

significant feature

of

the left side

of

Figure 4, a role can

alsobe envisioned forthe_right side, wherethe positively-charged Arg L55 is _{interacting through} a _bridging water molecule with the _{carboxylic acid group} on Sia-4. The negative chargeon _Asp

_HI05,

locatedatthebottomof the groove where it can form a_{hydrogen bond} to an _A-acetyl

group, mayalsoserve to directtheorientation

of

_{the hapten}

by repulsing its carboxylic acid _groups.

In all, theextended_{helical model proposed by Brisson}et al. (1992) with n = _6residues

per turn and_pitchofn x h

= ₃₆ A _{has a}

shape and charge distribution that is complementarytotheFabfrom_mAb735,andcan beshown

to form a _significantnumber

of

_specific contacts with the

Fabover a_{span at least}8sialic acidresiduesin_{length. This}

is close to the 10 residues predicted from earlier binding studies _(Hayrinen et al., 1989) and is confirmed by our

thermodynamic analysis.

CONCLUSIONS

X-ray crystallographic, thermodynamic, and NMR

evi-dence are consistent in the _prediction

of

a _binding site on

mAb735 which is at least eight residues in _{length. The}

crystallographic study is also consistent with the earlier prediction

of

a helical nature forthe _antigenic form

of

the oligosaccharide and provides a molecular basis for the recognition

of

the _{A-acetylneuraminic} acid. Trials are

underway to _{grow crystals}

of

the Fab _complexed with

oligosaccharides

of

different lengths; however, no _crystals

have beenobtained to date.

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